Liposome delivery systems have been proposed for a variety of drugs. For use in drug delivery via the bloodstream, liposomes have the potential of providing a controlled "depot" release of a liposome-entrapped drug over an extended time period, and of reducing toxic side effects of the drug, by limiting the concentration of free drug in the bloodstream. Liposome/drug compositions can also increase the convenience of therapy by allowing higher drug dosage and less frequent drug administration. Liposome drug delivery systems are reviewed generally in Poznansky et al.
One limitation of intravenous liposome drug delivery which has been recognized for many years is the rapid uptake of blood-circulating liposomes by the mononuclear phagocytic system (MPS), also referred to as the reticuloendothelial system (RES). This system, which consists of the circulating macrophages and the fixed macrophages of the liver (Kupffer cells), spleen, lungs, and bone marrow, removes foreign particulate matter, including liposomes, from blood circulation with a half life on the order of minutes (Saba). Liposomes, one of the most extensively investigated particulate drug carriers, are removed from circulation primarily by Kupffer cells of the liver and to a lesser extent by other macrophage populations.
A variety of studies on factors which effect liposome uptake by the RES have been reported. Early experiments, using heterogeneous preparations of multiamellar liposomes (MLV) containing phosphatidylcholine (PC) and cholesterol (CH) as their principal lipid constituents, demonstrated that these liposomes are rapidly removed from circulation by uptake into liver and spleen in a biphasic process with an initial rapid uptake followed by a slow phase of uptake (Gregoriadis, 1974; Jonah; Gregoriadis, 1972; Juliano). Half-time for removal of MLV from circulation was on the order of 5-15 min. following intraveneous (IV) injection. Negatively charged liposomes are removed more rapidly from circulation than neutral or positively charged liposomes. Small unilamellar liposomes (SUV) are cleared with half-lives approximately three-to four-fold slower than MLV (Juliano; Allen, 1983). Uptake of liposomes by liver and spleen occurs at similar rates in several species, including mouse, rat, monkey, and human (Gregoriadis, 1974; Jonah; Kimelberg, 1976; Juliano; Richardson; Lopez-Berestein).
Liposomes which are capable of evading the RES would have two important benefits. One is the increased liposome circulation time in the blood, which would both increase the pharmacokinetic benefits of slow drug release in the bloodstream, and also provide greater opportunity for tissue targeting where the liver, spleen, and lungs are not involved. The second benefit is decreased liposome loading of the RES. In addition to the role of the RES in removing foreign particles, the RES is involved in several other functions, including host defense against pathogenic microorganisms, parasites, and tumor cells, host responses to endotoxins and hemorragic shock, drug response, and responses to circulating immune complexes (Saba, Altura). It is important, therefore, in liposome administration via the bloodstream, to avoid compromising the RES seriously, by massive short-term or accumulated liposome uptake.
One approach which has been proposed is to increase liposome circulation time by increasing liposome stability in serum. This approach is based on studies by one of the inventors and others which have shown that factors which decrease leakage of liposome contents in plasma also decrease the rate of uptake of liposomes by the RES (Allen, 1983; Gregoriadis, 1980; Allen, 1981; Senior, 1982). The most important factor contributing to this effect appears to be bilayer rigidity, which renders the liposomes more resistant to the destabilizing effects of serum components, in particular high density lipoproteins (Allen, 1981; Scherphof). Thus, inclusion of cholesterol in the liposomal bilayer can reduce the rate of uptake by the RES (Gregoriadis, 1980; Hwang; Patel, 1983; Senior, 1985), and solid liposomes such as those composed of distearoylphosphatidylcholine (DSPC) or containing large amounts of sphingomyelin (SM) show decreased rate and extent of uptake into liver (Allen, 1983; Ellens; Senior, 1982; Hwang).
However, this approach appears to have a limited potential for increasing liposome circulation times in the bloodstream. Studies carried out in support of the present invention, and reported below, indicated that 0.1-0.2 micron liposomes containing optimal membrane-rigidifying liposome formulation are predominantly localized in the RES two hours after intravenous liposome administration. Although longer circulation times are achieved with small unilamellar vesicles or SUVs (having a size range between about 0.03 and 0.08 microns), SUVs are generally less useful in drug delivery due to their smaller drug-carrying capacity, and their greater instability, which can lead to rapid release of liposome-associated drug, and to liposome fusion events that produce large and heterogeneous-size liposomes.
Several investigators, including the applicants, have also explored the possibility of increasing liposome circulation times by designing the liposome surface to mimic that of red blood cells. The role of cell surface carbohydrates in cellular recognition phenomena is widely appreciated (Ashwell, Hakormori, Karlsson). The chemistry, metabolism, and biological functions of sialic acid have been reviewed (Schauer). Surface sialic acid, which is carried by gangliosides, and glycoproteins such as glycophorin, plays an important role in the survival of erythrocytes, thrombocytes, and lymphocytes in circulation. Enzymatic removal of sialic acid, which exposes terminal galactose residues, results in rapid removal of erythrocytes from circulation, and uptake into Kupffer cells of the liver (Durocher). Desialylation of thrombocytes (Greenberg) and lymphocytes (Woodruff) also results in their rapid removal by the liver.
Although desialylated erthrocytes will bind to Kupffer cells or peritoneal macrophages in vitro in the absence of serum, serum must be added in order for significant phagocytosis to occur. The nature of the serum components mediating endocytosis is speculative, but immunoglobin and complement (C3b) are thought to be involved. Czop et al. (Czop) have shown that sheep erythrocytes, which are not normally phagocytosed by human monocytes, will bind C3b and be phagocytosed upon desialylation. Okada et al. (Okada) have demonstrated that sialyglycolipids on liposome membranes restrict activation of the alternative complement pathway and that removal of the terminal sialic acid from the glycolipids abolishes this restricting capacity and results in activation of the alternative complement pathway. Sialic acid, therefore, may be functioning as a non-recognition molecule on cell membranes partly through its ability to prevent binding of C3b, thus preventing phagocytosis via the alternative complement pathway. Other immune factors may also be involved in liposome phagocytosis. Alving has reported that 50% of the test sera from individual humans contain naturally occurring "anti-liposome" antibodies which mediated complement-dependent immune damage to liposomes.
The observations reported above suggest that surface sialic acid, and/or other red-cell surface agents, incorporated into liposomes, for example, in the form of ganglioside or glycophorin, may lead to increased circulation half-lives of liposomes. This approach is described, for example, in U.S. Pat. No. 4,501,728 for "Masking of Liposomes from RES Recognition", although this patent does not disclose whether significant RES making is actually achieved by coating liposomes with sialic acid.
In fact, experiments conducted in support of the present applications indicate that sialic acid, in the form of gangliosides, has a limited ability to extend circulation half lives in vivo in liposomes which are predominantly composed of conventional liposomes lipids, such as egg phosphatidylcholine (egg PC) or egg PC:cholesterol mixtures. In vivo uptake studies on PC:cholesterol:ganglioside liposomes (0.2 microns or smaller) indicate that the injected liposomes are localized predominantly in the RES two hours post administration.
In summary, several approaches for achieving enhanced liposome circulation times in the bloodstream have been proposed. Heretofore, however, the approaches have produced quite limited improvements in blood circulation times, particularly in liposomes in the 0.07-0.2 micron size range which are generally most desirable for parenteral drug compositions.